Method and composition for inhibiting the growth of microorganisms



United States Patent METHOD AND COMPOSITION FOR INHIBITIN G THE GROWTH OF MICROORGANISMS Joseph Winkler, New York, N. Y., assignor to Fumol Corporation, a corporation of New York No Drawing. Application April 13, 1950, Serial No. 155,776

7 Claims. c1. 99-222 Dehydroacetic acid, which has the structure formula is also known as 3-acetyl-6-methyl pyrandione-2,4 and exists in both a keto and an enolic form. Dehydroacetic acid and its edible sodium, potassium, ammonium and calcium salts are known as non-hazardous preservatives to humans at used concentrations in food and beverages. Up to one tenth of a percent when incorporated into the beverage or foodstufi is almost undetectible by taste or smell. They are essentially neutral compounds and do not affect materially the pH of the foodstuff in which they are employed for protection against microorganism decay.

Table 1 shows concentration of dehydroacetic acid and/or its edible salts which still permit growth, as well as minimum concentrations which inhibit growth of different yeasts, fungi and bacteria. A careful study of table 1, however, brings one to the conclusion that depending upon the species of the microorganism, the minimum concentration of dehydroacetic acid or its salts (having an equal activity), varies in a very wide range; from 005% to .4%or differs about 80 times. A very high concentration of .3% to .4% is generally required to inhibit growth of bacteria, while on the ice TABLE 1 TESTS MADE WITH 100% DEHYDROACE'IIC ACID OR ITS EDIBLE SALTS Concen- Concen- (lt ratlont) 1trationt ercen ercen Test Organism Perm1t That ting Inhibits Growth Growth Aerobacter aerogenes 0. 2 0. 3 Alkaligeues faecalzs. 0. 3 0. 4 Aspergillus m'ger. 0. 03 0. 04 Bacillus cereus 0. 2 0. 3 Bacillus megatherium 0.2 0.3 Bacillus meseutericus... 0.2 0.3 Bacillus subtilis 0.2 0.3 Escherichia coli 0.3 0. 4 Lactobacillus plauatarium. 0. 08 0. 1 Penicillium digitatum.. 0.02 0.03 Je'aicillium ezpausum 0. 008 0.01 Pseudomonas aeruginosa. 0.3 0. 4 Rhzsopus nigricans 0. 04 0.05 Saccharomyccs cerevisiaa 0.05 0.1 Salmonella pullorum. 0.2 0.3 Salmonella typhose 0. 1 0.2 Staphylococcus aureus 0.2 0.3 Streptococcus pyugeues 0. 2 0.3 Trichophyton meutagrophytes 0. 004 0.005 Trichophyton interdigitale 0. 001 0. 005

Test period: 7 days for yeast and bacteria, two weeks for fungi.

Such an excessive amount of dehydroacetic acid, however, when added to foodstuff or beverages is sufficient so that it involves some detection of the chemical by taste. Such excessive amount also is not advisable from the economic point of view. Besides this, it may already involve some health hazards in prolonged sytematic consumption of the food preserved with such excessive amount. Because of the high cost of the material it is therefore highly desirable to keep said acid concentration in foodstuffs and beverages not higher than .1%, but at the same time to have proper protection against all kinds of microbial decay.

TABLE 1a TESTS MADE WITH 100% DEHYDROACETIC ACID IN COMPARISON WITH BA-Benzoic acid 100% SASalieylic acid 100% BHB-n-butylparahydroxybenzoate 100% D ChSDimethyldiehlorosuccinate 100% A.C oncentration permitting growth m w. percent X.Concentration that inhibits growth 111 percent 100% Dehydroacetie BA SA BHB D ChS Test organism Acld A. X. A. X. A. X. A. X. A. X.

Aspergillus uiger 03 05 65 75 l0 15 08 10 Bacillus mesenterz'cus .20 .30 50 75 50 60 10 .15 .08 .10 Escherichia coli 30 40 50 75 50 10 15 .08 10 Lactubacillus plautarium.-- 08 40 40 50 40 50 10 12 05 07 Penicillium ezpansum .008 01 15 .25 .20 30 l0 15 .05 06 Perticz'llium digitatu'm. 02 .03 40 50 30 40 l0 20 05 .06 Rhz'sopus m'grz'c'aus 04 05 40 50 30 40 08 10 08 10 Saccharomyces cerevisiae .05 10 40 70 30 40 20 30 .08 10 1 Test period: 7 days for yeast and bacteria, two weeks for fungi.

As the result of -extensive investigation in preparing compounds .which .could .increase the .anti=microbial activity of dehydroacetic acid or its edible salts, I have discovered that two classes of chemicals (which in themselves already possess in various but inferior degrees anti-microbial properties, with respect to dehydroacetic), are surprisingly highly efiective synergists for the antimicrobial activity of dehydroacetic acid or its edible salts. Their presence in quantities up to 50% of the total preservative concentration allows keeping the necessary minimum amount of the preservative composed predominantly of dehydroacetic acid or its edible salts, in alleases much lower than it wouldfollow from the additive antimicrobial power alone. See i. e. Table 1a, where the anti-microbial properties of dehydroacetic acid and some of my basic synergists are compared. Also in all cases thetnecessary minimum amount to give a omplete protect n to the .food ag inst m r organisms is not higher than .l%, or lies below the limits of government regulations for the addition of preservatives to foods and beverages.

I have found two definite classes of chemicals which act to greatly increase the anti-microbial properties of dehydroacetic acid or its edible salts.

4 percent concentration (by weight) necessary for inhibit- .inggrowth .of the specific microorganisms. The compounds listed in Table 2 are composed as follows:

A0 100% of dehydroacetic acid or its edible salt.

A1 80% A0 plus benzoic acid.

A2 70% A0 plus sodium benzoate.

A3 75% A0 plus 25% calcium benzoate.

A4 75% Au plus 25% salicylic acid.

A5 72% A0 plus 28% sodium salicylate.

As 65% A0 plus n-butylparahydroxybenzoate.

A7 80% A0 plus 20% n-propylparahydroxybenzoate.

As 83% A0 plus 17% ethylparahydroxybenzoate.

A9 70% A0 plus 30% sodiumpropylparahydroxy benzoate.

A10 50% A0 plus 50% dimethyldichlorosuccinate.

A11 55% Ag plus diisopropyldichlorosuccinate.

A12 55% A0 plus 35% diethyldichlorosuccinatc plus 10% benzoic acid.

A13 55% A0 plus 35% dimethyldichlorosuccinatc TABLE 2 Minimum quantities in weight per cent inhibiting growth (TESTSMADEIN VITRIO AS DONE IN TABLES 1 AND la Test, organism A0 A1 A2 As A4 A5 A9 A7 Ag A9 A 0 A 1 A 1 A 3 Aerobacter aerotmes 3 1 .1 l 09 .06 06 10.6 .06 0,6 .05 0a 0.: 05 Alkalige'nea laecalis. 4 09 O9 1 09 07 07 :07 .06 07 .06 06 06 05 Aspargillus uiger-" 04 02 03 03 03 O2 02 02 02 02 02 02 02 ()2 Bacillus cereus 3 09 09 08 08 08 07 07 07 .06 .06 07 06 05 Bacillus megathenum... 2 08 08 09 07 08 08 .07 .07 07 .06 07 06 05 BtZCtll'lLS meaeutencus- 3 08 07 07 08 07 07 07 .07 07 06 06 06 0. Bacillus subtilis. 4 08 08 07 06 06 06 .05 05 05 05 05 05 05 Each 'richia culi 3 O8 08 06 07 06 O5 05 05 07 06 05 05 05 Laclc bacillus planeta us 11 l0 09 08 09 07 06 05 06 08 05 06 05 05 Pam: zllium digitalum 03 02 03 03 02 Q1 ()1 Q1 01 01 Ol Pem illium azpansum- .01 .01 .01 01 01 005 005 .005 .005 005 005 005 005 Pteu domonas aeruginam .4 .085 1D 09 .09 08 08 O8 .07 09 06 06 05 05 Rhisopus niqricaus 05 03 03 02 02 03 03 02 02 03 02 02 02 02 Saccharomyces-ccrevisiaa 1 05 05 05 06 06 06 05 07 O7 05 04 04 0a Salmonella pullout/am.- 3 l 1 09 06 08 08 09 07 07 08 06 05 05 Staphylococcusaureus. '3 08 09 09 08 08 07 08 09 07 06 06 06 05 Streptococcus puqgeu es 3 09 09 08 09 08 09 09 08 06 06 06 06 06 Trichophyton 'meutagraphutes- 005 004 003 003 003 003 003 002 002 002 002 002 002 002 El'ipllophytauein terdigitalia 005 004 005i 003 003 003 003 003 002 002 002 002 002 002 l s p rlq 7 mus er y a t an b te a, 2 ee for un One cl ri e rom be zo c ac and ompr s s; en oic c d. it ibl ca i m. Po ssium, ammonium and sodium salts, its alkyl esters; andorthoand parah d oxy e oic acid a d t edibl s ts an a kyl es I e. f x mple an inc mplet is g o individual Compounds, as toll s; b nzo a id. s dium benz a potassium benzoate, calcium benzoate, ammonium benzoate, ethyl benzoate, salicylic acid, sodium salicylate, calcium salicylate, salicylic acid acetate, salicylic acid ethyl ester, para-hydroxymethylbenzoate, parahydroxyethylbenzoate, parahydroxypropylbenzoate'; n-butylparahydroxybenzoate, sodium salt of n-butylparahydroxybenzoate.

The other class is alkyl (Cl-C4) derivatives of alpha, beta-dichlor-succinic acid such as for example dimethyldichlorosuccinate, diethyldichlorosuccinate, dipropyldichlorosuccinate, diisopropyldischlorosuccinate, dibutyldichlorosuccinate.

A mixture consisting of up to of dehydroacetic acid or its edible salt and the balance being one or more of the above mentioned activating compounds, has a highly balanced anti-microbial activity, and always in amounts below .1% acts as .a universal preservative against all classes of microorganisms. The results obtained with some of the activated compounds are compared with dehydroacetic acid or its edible salts in Table 2. The preservatives are there given with code names from A0 to A13, and the quantifies are minimum From the results given on table 2 it follows that any f he mat ri l dentified a 1 till A13 is sup to a high degree over dehydroacetic acid alone and even for the most resistant microorganisms the required minimum concentration inhibiting growth is below .1%. This is a highly defiirable result, allowing the preservative compositions to be used sparingly in food preservation. Practical application for food and beverage preservation of compounds made according to the present invention have shown that the necessary amount was even much lower than .1%,'and under same conditions at least half as much aswith dehydroacetic acid or its edible salts alone.

Table 3 gives further evidence and examples of the preserving qualities of some of the activated compounds of dehydroacetic acid in accordance with this invention, applied to dififerent beverages and foodstuffs either when incorporated directly into the food or applied as a wash, spray or dip, or when incorporated into the wrapper. These tests have been conducted upon the same samples of the beverages and foodstuffs, applying the same conditions of time, temperature and humidity. The indi cated amounts of the preservative compounds are minimum quantities necessary to prevent spoilage from the inherent microorganisms in the particular beverage or is normally only a fraction of the weight of the food, even 3. A composition as claimed in claim 1, in which the in the unlikely event of a complete transfer of preservative synergist is n-butylparahydroxybenzoate.

to food, the concentration in the food will be substantially 4. A composition as claimed in claim 1, in which the below .I%. synergist is propylparahydroxybenzoate.

Furthermore, in Table No. 3, where a wash, dip or 5 5. A composition as claimed in claim spray is used, the figures given indicate the concentration synergist is ethylparahydroxybenzoate.

of the preservative in the solution used as the wash, dip or 6. A composition as claimed in claim 1, in which the 1, in which the spray. synergist is sodium propylparahydroxybenzoate.

TABLE 3 Necessary Minimum Concentration in Weight Percent S Tested Beverage or Foodstufl Mode of Application A A: At A10 1. Beer Dissolved .05 .03 .02 Not applied. 2. Wine do .07.1 .04.06 .04-.05 Do. 3. Apple juice. do .03.5 .02.04 .02-.03 D0. 4. Orange juice do .02-. 01-. 02 01-.02 Do. 5. Corn syrup do .03. 5 01-. 02 01-. 02 Do. 6. Chocolade "hot fudge syrup do .024-.5 .01.02 01-. 015 Do. 7. Milk Incorporated into the wall of 1-.2 .05-.1 .03-.07 Do.

the container paper board. 8. Whole bread Added to the dough 1-. 2 05- 1 03-. 07 Do. 8a. Whole bread Incorporated into the bread 5-.2 5 .3-.4 .2-.3.

wrapper, paper, glassine, cellophane. 9. Gingerbread Added to the dough 075 05 04-.05 Not applied. 9a. Gingerbread Incorporated into the wrapper, 5-. 2 3- 5 3-.4 .2-. 3.

paper, glassine, cellophane. 10. Cream cheese Added to thecheese 1 .04-.06 .03-.04 Not Applied. 10a. Cream cheese- Incorporated into the wrapper 5-1 2-. 5 2- 4 2- 3.

or container. 11. Frankfurters Added to themeat .2 1 .05. 1 Not Applied. 11a. Frankfurters Incorporatedmtothewrapper, ,5-1 ,2-.3 2- 3 .1-. 2.

- paper, glassine, cellophane,

or casing. 12. Smoked meat, bacon, ham Dippedorsprayedconaofthe 1-.2 .04. 05 .04. 05 .03 .05.

solution, weight percent. 12a. Smoked meat, bacon, ham lncorporatedmt thewrapper, 5 .2. 3 2*. .15. 2.

i. e. paper, casing, glassine, cellophane, plastic coating. 13. Edible tat, butter, lard, margarine, short- Incorporated into the [at .05 ,02-. 03 ,015- 025 Not Applied,

enmg, mayonaise. 13a. Edible fat, butter, lard, margarine, short- Incorporatedmtothewrapper, 5 2-. 3 20 15-. 20,

ening, mayonaise. 1. 9. paper glassrne, etc. 14. Tobacco, chewing tobacco, cigarettes, Incorporated into the wrapper. .5 .2-.3 .2-.3 1-. 2.

cigars, etc. 14a. Tobactco, chewing tobacco, cigarettes, Sprayed .4 .2-.3 .1-.2 .1. 2.

cigars, e c. 15. Dried fruits Sprayed Cone of the solution.. .1-.3 .05-.06 .05-.06 .04-. 05. 15a. Dried fruits lncorporatedrntothewrapper, .5 2.3 .2-.3 -.z.

i. e. paper, cartonboard, boxes, etc. 16. Fresh berries strawberries, raspberries, Washed. Cone .1 .05 .05 .04-.05.

blackberries, cherries, grapes, etc. 16a. Fresh berries, strawberries, raspberries, Incorporated mto the pack- .5 .2-.3 ,2-.3 .1-. 2.

blackberries, cherries, grapes, etc. aging (Paper, boxes, etc.) 17. Citrus fruits, apples, pears, avocados, etc..- Washed, drplped or sprayed, ,2 .1 .1 .1.

Cone. of t e solution in wt. percent. 17a. Citrus fruits, apples, pears, avocados, etc.. Incorporatedintothewrapper, 5 2-.3 .2-.3 .1-.2

paper, cellophane, limngs, etc. 18. Cantaloupes, melons, bananas, tomatoes-.. Washed, sprayed, nipped, 1 .05 .05 .05.

come. of thesolutron. 18a. Cantaloupes, melons, bananas, tomatoes Incorporatedmtothewrapper, ,5 .2-.3 .2-.3 .1-,2,

Paper, cellophane, liner, etc. 19. Vegetables, cucumbers, potatoes, carrots, Washed, s rayed, d pped, 2 .1 .1 .1.

beets, etc. Cone. gt t e solution m wt.

percen 19a. Vegetables, members, potatoes, carrots, Incorporatedintothewrapper, .5 2-.3 .2-.3 1-.2

beets, etc. liaper, lmmg, bags, boxes,

e c. 20. Chocolate filled candies, etc Incorporatedintothewrapper, 5 2-.3 .2-.3 1-. 2.

paper, cellophane, foil, etc.

While, for the purpose of illustration, the present invention is described in detail in connection with foodstuffs and other edible substances, it is also applicable where it is desired to inhibit, in a manner non-toxic and non-irritating to humans and warm blooded animals, microorganisms in articles or substances, as for example, cosmetics, lotions, ointments, handkerchiefs, towels, napkins, bandages, dressings, absorbent cotton, etc.

I claim:

1. A composition of matter for inhibiting microorganisms, comprising as preserving agent a mixture of 83% to by weight of a dehydroacetic acid compound selected from the group consisting of dehydroacetic acid and its edible salts with 17% to 50% by weight of a synergist consisting of a benzoic acid compound selected from the group consisting of benzoic acid and its edible derivatives.

2. A composition as claimed in claim 1, in which the synergist is benzoic acid.

7. A method of inhibiting microorganisms in a substance which supports microorganic growth, comprising References Cited in the file of this patent UNITED STATES PATENTS 1,627,342 Sabalitschka May 3, 1927 1,715,251 Sabalitschka et a1. May 28, 1929 2,474,228 Coleman ct al. June 28, 1949 2,480,010 Flctt Aug. 23, 1949 

1. A COMPOSITION OF MATTER FOR INHIBITING MICROORGANISMS, COMPRISING AS PRESERVING AGENT A MIXURE OF 83% TO 50% BY WEIGHT OF A DEHYDROACETIC ACID COMPOUND SELECTED FROM THE GROUP CONSISTING OF DEHUDROACETIC ACID AND ITS EDIBLE SALTS WITH 17% TO 50% BY WEIGHT OF A SYNERGIST CONISTING OF A BENZOIC ACID COMPOUND SELECTED FROM THE GROUP CONSISTING OF BENZOIC ACID AND ITS EDIBLE DERIVATIVES. 